Maturation of Purkinje cell firing properties relies on neurogenesis of excitatory neurons
- Department of Pathology and Immunology, Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, United States
- Department of Neuroscience, Baylor College of Medicine, United States
- Program in Developmental Biology, Baylor College of Medicine, United States
- Howard Hughes Medical Institute, Department of Molecular and Human Genetics, Baylor College of Medicine, United States
- Development, Disease Models and Therapeutics Graduate Program, Baylor College of Medicine, United States
Figures
Figure 1 with 4 supplements
Conditional deletion of Atoh1 from the En1 domain prevents proper size expansion, lamination, and foliation of the cerebellum.
(A) Schematic of an embryonic brain. Inset is the cerebellar anlage. Atoh1 domain (excitatory lineage neurons, including granule cell precursors, pink), Ptf1a domain (inhibitory lineage neurons, …
Figure 1—figure supplement 1
Conditional deletion of Atoh1 from the En1 domain leads to cerebellum-specific morphological abnormalities.
(A) Whole brain sagittal tissue sections from control mice stained using cresyl violet demonstrate the typical anatomical features found throughout the forebrain, midbrain and hindbrain. (B) Whole …
Figure 1—figure supplement 2
Conditional deletion of Atoh1 from the En1 domain reduces the density of excitatory cerebellar cell types, but increases the density of inhibitory cerebellar cell types.
(A) En1Cre/+;Atoh1fl/- mice lack differentiated granule cells, identified with GABARα6. (B and C) En1Cre/+;Atoh1fl/- mice have a reduction in unipolar brush cells, identified by Calretinin and Tbr2. …
Figure 1—figure supplement 3
Conditional deletion of Atoh1 from the En1 domain reduces the density of En1;Atoh1 lineage neurons.
(A) Genetic strategy to express TdTomato in the En1;Atoh1 lineage of control mice. (B) Genetic strategy to express TdTomato in the En1;Atoh1 lineage of conditional knockout mice. (C) Expression of …
Figure 1—figure supplement 4
Conditional deletion of Atoh1 from the En1 domain reduces the number of unipolar brush cells and excitatory nuclei neurons.
(A) Co-expression of Tbr2 (green) and TdTomato (pink) in the En1;Atoh1 lineage (as in Figure 1—figure supplement 3) in control (top) and conditional knockout (bottom) cerebellums. (B) Co-expression …
-
Figure 1—figure supplement 4—source data 1
Source data and specific p-values for representative graphs in Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig1-figsupp4-data1-v1.xlsx
Figure 2
Atoh1 lineage neurons are necessary for the formation of sharp zonal patterns of Purkinje cell sub-types and spinal cord mossy fiber projections.
(A) Top view of a control P14 brain. Dotted line shows the position of the schematic in (B) and where the section in (C) was taken from. (B) Schematic of Purkinje cell ZebrinII (pink) and PLCβ4 …
Figure 3
Atoh1 lineage neurons are necessary for establishing the dendritic complexity of Purkinje cells.
(A) Representative images of Golgi-Cox-labeled Purkinje cells in the cerebella of control (top row) and En1Cre/+;Atoh1fl/- (bottom row) mice. (B) Sholl analysis for dendritic complexity. (C) …
-
Figure 3—source data 1
Source data and specific p-values for representative graphs in Figure 3.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig3-data1-v1.xlsx
Figure 4
The density of Vglut1 synapses onto Purkinje cells increases between P7 and P14 in control mice and is lower in En1Cre/+;Atoh1fl/- mice.
(A) Schematic of Purkinje cell microcircuit in control (left) and En1Cre/+;Atoh1fl/- mice (right) at P7 and P14. Vglut1 is expressed in granule cell (pink) and some mossy fiber (gray) terminals but …
-
Figure 4—source data 1
Source data and specific p-values for representative graphs in Figure 4.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Purkinje cell simple spike firing patterns transform from P7 to P14 in control mice and reflect altered maturation in P14 En1Cre/+;Atoh1fl/- mice.
(A) Schematic of in vivo Purkinje cell recordings in P7 control mouse. Top left: cerebellum with recording electrode. Top right: schematic with recording electrode extracellular to Purkinje cell …
-
Figure 5—source data 1
Source data and specific p-values for representative graphs in Figures 5 and 6.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Simple spike distributions of single Purkinje cells.
Heatmap of frequency distribution of simple spike firing patterns in Purkinje cells of different ages in control and En1Cre/+;Atoh1fl/- mice.
Figure 6 with 1 supplement
Purkinje cell simple spike firing patterns undergo a developmental transformation between P7-P10 and P11-P14 that does not occur in En1Cre/+;Atoh1fl/- mice.
(A) Representative electrophysiological recordings (each 10 s long) from Purkinje cells recorded in P7-P14 control mice and P10 and P14 En1Cre/+;Atoh1fl/- mice. In some recordings, we observed …
Figure 6—figure supplement 1
Simple spike firing properties do not normalize by P18 in En1Cre/+;Atoh1fl/- mice.
(A) Simple spike firing frequency (spikes/recording time). (B) Simple spike frequency mode (peak ISI−1 distribution). (C) Pause percentage (proportion of recording with ISI > five times average …
Figure 7
The density of Vglut2 synapses onto Purkinje cells decreases between P7 and P14 in control mice and is higher in En1Cre/+;Atoh1fl/- mutants.
(A) Schematic of Purkinje cell microcircuit in control (left) and En1Cre/+;Atoh1fl/- mice (right) at P7 and P14. Vglut2 is expressed in climbing fibers (orange) and some mossy fibers (gray) but not …
-
Figure 7—source data 1
Source data and specific p-values for representative graphs in Figure 7.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig7-data1-v1.xlsx
Figure 8
Purkinje cell complex spike firing patterns change from P7 to P14 in control mice and are altered in P14 En1Cre/+;Atoh1fl/- mice.
(A) Schematic of in vivo Purkinje cell recordings in P7 control mouse. Top left: cerebellum with recording electrode. Top right: schematic showing the recording electrode extracellular and adjacent …
-
Figure 8—source data 1
Source data and specific p-values for representative graphs in Figure 8.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig8-data1-v1.xlsx
Figure 9
Postnatal En1Cre/+;Atoh1fl/- mice have abnormal motor coordination, enhanced tremor and altered vocalization behavior.
(A) Timed-series photos of Atoh1fl/+ (gray) and En1Cre/+;Atoh1fl/- (orange) mice. En1Cre/+;Atoh1fl/- mice have a wide stance (blue arrows) and fall on their backs (red arrows). (B) Time to right in …
-
Figure 9—source data 1
Source data and specific p-values for representative graphs in Figure 9.
- https://cdn.elifesciences.org/articles/68045/elife-68045-fig9-data1-v1.xlsx
Videos
Video 1
En1Cre/+;Atoh1fl/- mice have a spectrum of visible motor impairments.
At P14, control mice explore the open box smoothly, whereas En1Cre/+;Atoh1fl/- mice have a visible tremor, often fall on their backs, and have dystonia-like postures and movements in their hindlimbs.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Calbindin (guinea-pig polyclonal) | Synaptic Systems | RRID:AB_2619902 | IF: (1:1,000) |
| Antibody | Anti-GABARα6 (rabbit polyclonal) | Millipore Sigma | RRID:AB_91935 | IF: (1:500) |
| Antibody | Anti-Tbr2 (rabbit polyclonal) | Abcam | RRID:AB_778267 | IF: (1:500) |
| Antibody | Anti-Calretinin (mouse monoclonal) | Swant | RRID:AB_10000320 | IF: (1:500) |
| Antibody | Anti-NFH (mouse monoclonal) | Biolegend | RRID:AB_2564642 | IF: (1:1,000) |
| Antibody | Anti-HCN1 (rabbit polyclonal) | Alomone Lab | RRID:AB_2039900 | IF: (1:500) |
| Antibody | Anti-RORα (goat polyclonal) | Santa Cruz | RRID:AB_2301066 | IF: (1:250) |
| Antibody | Anti-PV (rabbit polyclonal) | Swant | RRID:AB_10000344 | IF: (1:1,000) |
| Antibody | Anti-neurogranin (rabbit polyclonal) | Millipore | RRID:AB_91937 | IF: (1:500) |
| Antibody | Anti-ZebrinII (mouse monoclonal) | Gift from Dr. Hawkes | IF: (1:500) | |
| Antibody | Anti-Vglut1 (rabbit polyclonal) | Synaptic Systems | RRID:AB_887877 | IF: (1:500) |
| Antibody | Anti-Vglut2 (rabbit polyclonal) | Synaptic Systems | RRID:AB_887883 | IF: (1:500) |
| Antibody | Anti-PLCβ4 (rabbit polyclonal) | Santa Cruz Biotechnology | RRID:AB_654082 | IF: (1:150) |
| Chemical compound, drug | WGA-Alexa 555 | Thermo Fisher Scientific | ||
| Chemical compound, drug | Fast green | Sigma-Aldrich | ||
| Chemical compound, drug | PBS | Sigma-Aldrich | ||
| Commercial assay or kit | Golgi-Cox staining kit | FD Neurotechnologies | ||
| Strain, strain background (M. musculus) | Mouse: Atoh1-FlpO | Gift from Dr. Zoghbi | van der Heijden and Zoghbi, 2018 | |
| Strain, strain background (M. musculus) | Mouse: Rosa-FSF-LSL-tdTomato (Ai65(RCFL-tdT), Gt(ROSA)26Sortm65.1(CAG-tdTomato)Hze) | The Jackson Laboratory | RRID:IMSR_JAX:021875 | |
| Strain, strain background (M. musculus) | Mouse: En1-Cre (En1tm2(cre)Wrst/J) | The Jackson Laboratory | RRID:IMSR_JAX:007916 | |
| Strain, strain background (M. musculus) | Mouse: Atoh1-Flox (Atoh1tm3Hzo) | The Jackson Laboratory | RRID:MGI:4420944 | |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | ImageJ | National Institutes of Health | RRID:SCR_003070 | |
| Software, algorithm | Photoshop | Adobe | RRID:SCR_014199 | |
| Software, algorithm | Illustrator | Adobe | RRID:SCR_010279 | |
| Software, algorithm | Spike2 | CED | RRID:SCR_010279 | |
| Other | Tungsten electrodes | Thomas Recording | ||
| Other | VetBond | 3M |
